ADP-ribosylating bacterial exotoxins are widely known. Examples include diphtheria toxin (Corynebacterium diphtheriae), exotoxin A (Pseudomonas aeruginosa), cholera toxin (CT; Vibrio cholerae), heat-labile enterotoxin (LT; E. coli) and pertussis toxin (PT).
The toxins catalyse the transfer of an ADP-ribose unit from NAD+ to a target protein. CT, for instance, transfers ADP-ribose to a specific arginine side chain of the α subunit of GS, which blocks the ability of Gs to hydrolyse GTP to GDP. This locks the protein in its ‘active’ form, so adenylate cyclase activity is permanently activated. Cellular cAMP levels rise, leading to the active transport of ions from the cell and the loss of water into the gut [1].
The toxins are typically divided into two functionally distinct domains—A and B. The A subunit is responsible for the toxic enzymatic activity, whereas the B subunit is responsible for cellular binding. The subunits might be domains on the same polypeptide chain, or might be separate polypeptide chains. The subunits may themselves be oligomers e.g. the A subunit of CT consists of A1 and A2 which are linked by a disulphide bond, and its B subunit is a homopentamer. Typically, initial contact with a target cell is mediated by the B subunit and then subunit A alone enters the cell.
Crystal structures [2] are known for LT [3], CT [4] and PT [5].
The toxins are typically immunogenic, and have been proposed for use in acellular vaccines. One problem, however, is that the proteins retain their toxic activity in the vaccines. To avoid this problem, site-directed mutagenesis of key active site residues has been used to remove toxic enzymatic activity whilst retaining immunogenicity [e.g. refs. 6 (CT and LT), 7 (PT), 8 etc.]. Current acellular whooping cough vaccines include a form of pertussis toxin with two amino acid substitutions (Arg9→Lys and Glu129→Gly; ‘PT-9K/129G’ [9]).
As well as their immunogenic properties, the toxins have been used as adjuvants. Parenteral adjuvanticity was first observed in 1972 [10] and mucosal adjuvanticity in 1984 [11]. It was surprisingly found in 1993 that the detoxified forms of the toxins retain adjuvanticity [12].
Although they display the same catalytic activity, the primary and secondary structures of ADP-ribosylating toxins are poorly conserved. Reference 13 discloses six ADP-ribosylating toxins with only a low level of sequence identity to toxins such as CT, LT and PT, from Neisseria meningitidis, Streptomyces coelicolor, Mycoplasma pneumoniae, Salmonella typhimurium, Salmonella paratyphi, and Streptococcus pyogenes. Mutants of the toxins are also disclosed.
It is an object of the invention to provide further mutant N. meningitidis toxins.
They are preferably prepared in substantially pure form (i.e. substantially free from host cell proteins).
The invention also provides the proteins of the invention for use as immunogens and/or as adjuvants and, in particular, as mucosal and/or parenteral adjuvants.
The invention also provides the use of proteins of the invention in the manufacture of a medicament for raising an immune response in an animal. The medicament is preferably an immunogenic composition (e.g. a vaccine), and may comprise, in addition to a protein of the invention, an antigen against which an enhanced immune response is to be raised. The medicament is preferably administered mucosally e.g. orally or intranasally.
The invention also provides immunogenic compositions (e.g. a vaccine) comprising a protein of the invention in admixture with a second antigen. It also provides a kit comprising a protein of the invention and a second antigen for simultaneous, separate or sequential administration. The second antigen is preferably one of the N. meningitidis proteins disclosed in references 15 to 21. The composition may comprise a third antigen, a fourth antigen, a fifth antigen etc., one or more of which may be selected from the N. meningitidis proteins disclosed in these seven references.
According to a further aspect, the invention provides antibody which binds to a protein of the invention. These may be polyclonal or monoclonal and may be produced by any suitable means. The antibody may include a detectable label. The antibody will bind to an epitope which includes one or more of amino acids Glu-109, Glu-111 or Glu-120.
According to a further aspect, the invention provides nucleic acid encoding the proteins of the invention. The invention includes nucleic acid comprising sequences complementary to those described above (e.g. for antisense or probing, or for use as primers).
Nucleic acid according to the invention can, of course, be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (e.g. single stranded, double stranded, vectors, primers, probes etc.).
Nucleic acid according to the invention may be labelled e.g. with a radioactive or fluorescent label. This is particularly useful where the nucleic acid is to be used as a primer or probe e.g. in PCR, LCR or TMA.
In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.
According to a further aspect, the invention provides vectors comprising nucleic acid of the invention (e.g. cloning or expression vectors) and host cells transformed with such vectors.